Aqueous extract of ground material of planarian organisms

ABSTRACT

The present invention relates to an aqueous extract useful notably for the regeneration of human or animal cells, notably mammalian cells, comprising an aqueous extract free of any insoluble solid debris of ground planarian organisms having a regeneration capacity, containing at least the intracellular components of the cells of said organisms of a cell lysate of said cells of said organisms.

The present invention relates to a preparation based on an extract ofPlatyhelminthes organisms useful notably for the regeneration of humanor mammalian animal cells.

The term “planarian” is used to refer to a number of flatworms of thephylum Platyhelminthes. It mainly refers to non-exclusively parasiticfreshwater Turbellaria, i.e. Paludicola, but is also used for marineTurbellaria and even for all flatworms. The Platyhelminthes areflatworms of which many species are parasites. This phylum consistsmainly of worms that are elongated animals without appendages. The bestknown worms of the class Turbellaria (flat worms that are notexclusively parasitic) are the planarians, worms whose body thicknesscan measure less than one millimetre. Planarians are aquatic flatwormsbelonging to several species of the class Turbellaria. Planarians canswim or crawl, and live in the sea or freshwater, or in very wet soil(in tropical forests).

Planarians are organisms capable of perpetual regeneration, and any partof their body, due to the presence of a large quantity of stem cells(20-30%) in their tissues in comparison with other living organisms,which have roughly 1 ppm stem cells. Planarian stem cells multiply andregenerate continuously, unlike stem cells in more evolved organisms,which multiply little if at all. The biochemical factors that allow stemcells to multiply in a planarian cell remain unidentified.

Because of its regenerative capacity, if one part of a planarian isseparated from the rest of its body, the animal recreates the othermissing part in its entirety. This phenomenon of “transverse fission”,also involved in asexual reproduction, exists in other animals,particularly metazoans, but few can compete with planarians in thisarea.

In the article by Bocchinfuso et al. in Molecular & Cellular Proteomicsvol 11 no 9, September 2012, pages 681 to 691, a non-aqueous proteinextract obtained by chemical lysis of mucous secretions of Schmidteamediterranea worms is described. In this article, only the treatment ofinfection and mucus disorders such as asthma and lung disease or cysticfibrosis is considered.

In the article by Changxin Ma et al in Journal of Genetics and Genomicsvol 37 no 11 of November 2010, pages 713-723, here again a non-aqueousprotein extract from the planarian species Dugesia japonica isdescribed. These organic extracts are prepared for the sole purpose ofproteomic study and identification of a gene involved in optic nerveregeneration.

In WO 99/33479, the therapeutic use for the treatment of autoimmunediseases of preparations based on cercariae, pathogenic parasitesnaturally colonizing humans and not exhibiting the property ofregenerating, is described, the preparations being obtained byrecovering parasite eggs from hamster liver to which said cercariae havebeen injected.

In the article in Hydrobiologia vol 305 no 1-3 of June 1995, pages247-253, an extract of Dugesia japonica obtained by recovering only theheads of the Dugesia japonica organisms concerned subjected to chemicaland sonication treatments and then dialysis to recover molecularextracts with a molecular size of less than 10,000 is described. In theend, only precipitated proteins are recovered. On the other hand, cellgrowth tests were performed on Neuro 2a and PC-12 cells. The reportedresults conclude that these protein extracts inhibit the growth of nervecells and even inhibit the regeneration of the heads of said decapitatedplanarian organisms, whereas in the absence of treatment with these ASPplanarian extracts regeneration is observed. It has been discovered,according to the present invention, that an aqueous extract of planarianflatworms, which are non-parasitic and non-pathogenic in humans oranimals, notably of the species Schmidtea mediterranea, is capable ofactivating the multiplication of human stem cells, which planarianextract, because of this cell regeneration activity, can therefore beuseful in many cosmetic or medical applications, in particulardermatological or food applications.

The expression “non-parasitic and non-pathogenic in humans or animals”,as used herein, means that such organisms do not colonize humans ormammalian animals either by ingestion or through the skin, eitheractively or passively, by pathologically altering human or mammaliananimal cells or tissues.

More precisely, the present invention provides an aqueous extract usefulnotably for the regeneration of human or mammalian animal cells,comprising an aqueous extract free of any insoluble solid debris ofground organisms of the Platyhelminthes type having a regenerativecapacity, said organisms being aquatic flatworms of the planarian typewhich are non-parasitic and non-pathogenic in humans or mammaliananimals, said aqueous extract containing at least the intracellularcomponents of the cells of said organisms of a cell lysate of said cellsof said whole organisms.

The term “cell lysate”, as used herein, means that the cells of saidorganisms are broken down mechanically so that intracellular componentsare released into said extract when said organism is ground. And, saidaqueous extract of ground material is an aqueous extract of said groundorganisms free of any insoluble solid debris, i.e., only retained is the“planarian liquor” resulting from mechanical grinding containing onlythe intracellular components of the cells of said organisms of a celllysate of said cells of said organisms, but all components and not onlypeptides and proteins.

More particularly, said planarian type aquatic flatworms that arenon-parasitic and non-pathogenic in humans include Schmidteamediterranea, Dugesia japonica, Dendricoelum lacteum, Polycelis nigra,Polycelys tenuis or Planana torva.

More particularly still, said aqueous extract of ground materialcomprises a centrifugation supernatant of said ground material.

More particularly still, said aqueous extract of ground material furthercomprises a cell culture medium, preferably a culture medium capable ofculturing human or animal cells to be regenerated.

More particularly still, said aqueous extract of ground materialcomprises the grinding of 50 to 200 mg worms/mL aqueous solution, inparticular for worms of 1 to 5 mg, said aqueous extract of groundmaterial being further diluted preferably more than 1:5 and less than1:100, preferably diluted 1:10 to 1:50; notably 1:20, preferably stilldiluted in said culture medium of said cells.

In particular, for worms of 2.2 mg on average, said aqueous extract ofground material comprises the ground material of 88 to 110 mg worms/mLaqueous solution, i.e. 40 to 50 worms/mL, said aqueous extract of groundmaterial being further diluted 1:20.

More particularly still, a spectrum of said aqueous extract of groundmaterial by MALDI-TOF mass spectrometry comprises the 16 majoritycharacteristic peaks (i.e. higher intensities(maximum S/N value)mentioned in the following Table 1, in which the various characteristicpeaks are numbered in the first column, and the following parametershave the following meanings:

M/z: ratios (mass in daltons/charge) on the x-axis of the differentcharacteristic peaks of the spectrum,

S/N: ratios (peak intensity/background noise intensity) of the differentcharacteristic peaks of the spectrum,

TABLE 1 M/Z S/N standard standard M/Z deviation S/N deviation 1 8618.401118.031 10852424.610 11358052.088 2 8270.340 143.008 4620996.2745564954.057 3 7696.982 133.512 5240359.923 7010776.551 4 7236.251151.442 8121705.361 11556323.264 5 6803.343 116.896 10708147.03418373045.879 6 6261.480 163.376 11319617.938 13604702.931 7 5790.573116.345 21402383.693 37624416.818 8 5330.950 180.245 23085485.04921438811.717 9 4787.963 120.365 22731248.668 23171439.149 10 4298.636175.285 24470357.501 21953618.321 11 3793.559 113.425 21664847.04517673326.341 12 3282.861 172.508 24634530.736 27003268.283 13 2792.441122.205 22551669.576 22432014.766 14 2294.281 174.311 20589324.29917568023.101 15 1807.544 115.976 19890585.114 18182405.582 16 1395.545119.208 33019669.177 29193485.572

The present invention also provides a lyophilisate of an aqueous extractof ground material according to the invention.

The present invention also provides a process for producing an aqueousextract of ground material according to the invention characterized inthat the steps are carried out in which:

a) said living organisms, preferably which have been washed with waterand have not been fed for at least one week, are placed in an aqueousliquid medium containing preferably a cell culture medium;

b) the aqueous preparation obtained in step a) is ground, notably bymechanical grinding, until lysis of the cells of said organisms isobtained, preferably the ground material debris being between 5 and 10μm in size (i.e. debris smaller than the size of the cells of saidorganisms);

c) insoluble debris is removed by centrifugation; and the supernatant iscollected,

d) preferably, the centrifugation supernatant is diluted in an aqueoussolution more than 1:5 and less than 1:100, preferably diluted between1:10 and 1:50, said solution comprising also preferably components of acell culture medium capable of culturing human or animal cells to beregenerated.

The present invention also relates to the use of an aqueous extract ofground material or lyophilisate according to the invention for themanufacture of a composition useful for regenerating human or mammaliananimal cells, notably stem or differentiated cells.

In particular, mention may be made of differentiated cells of thefollowing types: epithelial, fibroblast, keratinocyte, endothelial,neuronal or nerve.

The present invention also relates to the use of an aqueous extract ofground material or lyophilisate according to the invention for themanufacture of a composition useful for regenerating human or mammaliananimal stem cells, for example a composition useful for regeneratinghuman dental stem cells.

The present invention also relates to the use of an aqueous extract ofground material or lyophilisate according to the invention for themanufacture of a cosmetic or dermatological composition in combinationwith suitable carriers and/or excipients, notably an anti-ageingcosmetic or dermatological composition formulated for topicalapplication in the form of gel, lotion (notably an aqueous solution orserum), cream, ointment, soap and paste (notably for a mask).

The present invention also relates to the use of an aqueous extract ofground material or lyophilisate according to the invention for themanufacture of a food composition.

More particularly still, an anti-ageing cosmetic product according tothe invention is formulated for topical application in the form of gel,lotion, cream, ointment, soap or paste.

The topical compositions according to the invention may comprise varioususual excipients suitable for external topical administration, inparticular dermatologically and cosmetically acceptable excipients.These excipients suitable for formulation are well known to the skilledperson and include, for example, penetrants; moisturizers; thickeners;emollients and surfactants; emulsifiers; preservatives.

These forms of topical administration are prepared by known techniques,and for example, in the case of a cream, by dispersing a fatty phase inan aqueous phase to obtain an oil-in-water emulsion, or conversely toprepare a water-in-oil emulsion.

In a particular embodiment, the cosmetic product according to theinvention is formulated in the form of cream comprising, said aqueousextract and at least the following additional excipients: emulsifier,emollient, thickener, moisturizer and preservatives and pH adjuster.

In a particular embodiment, the cosmetic product according to theinvention is formulated in the form of aqueous solution (notablyphysiological water serum) or of aqueous gel comprising, as ahomogeneous mixture in water, said aqueous extract and, for the gel, atleast the following additional gel excipients: gelling agent, thickener,moisturizer and preservative and pH adjuster.

The preparations generally contain other excipients and/or auxiliariesand sometimes other active principles, e.g. dyes, colouring pigments,radical scavengers, fragrances, stabilizers.

The cosmetic products may also include auxiliaries and sometimes otheractive principles, for example antioxidant vitamins such as vitamin E,vitamin C, antioxidant agents such as natural polyphenols, enzymes,plant active principles, natural anti-inflammatory substances, alcohols,polyols, esters, electrolytes, polar and non-polar oils, polymers,copolymers, phospholipids, dyes; fragrances; or skin scrubs.

The invention further relates to a cosmetic skin treatment process forcombating the signs of skin ageing, and in particular expressionwrinkles caused by uncontrolled facial muscle contractions, consistingin applying to the areas of the skin requiring such treatment, a topicalcosmetic product according to the invention.

The present invention therefore relates to a cosmetic composition withan anti-ageing effect on the skin comprising an aqueous or lyophilizedextract according to the invention and excipients suitable for topicalapplication to the skin in the form of gel, lotion, cream, ointment orsoap.

Other features and advantages of the present invention will becomeclearer by reading the description of the examples that follow, which isillustrative and non-limiting, with reference to the following figures.

FIGS. 1A to and 1C represent the growth on day 9 of human dental stemcells treated with planarian extracts from Example 3A diluted 1:20 (FIG.1A: aqueous extract (A1), extract containing PBS (A2) and extractscontaining cell culture medium (A3) diluted 1:5 (FIG. 1B) and 1:100(FIG. 1C).

FIG. 2 represents microphotographs showing the growth of human stemcells treated with extract A diluted 1:20 for 9 days (B2) and beforetreatment (B1). The cells are stained with Giemsa.

FIG. 3 shows the lack of growth on day 9 of human cells treated withground whole planarians not free of solid debris of Example 1.

FIG. 4 shows the growth of human cells treated with the solubilizedlyophilized extract of Example 3B.

1—METHODS

1.1 Planarians:

Planarians of the species Schmidtea mediterranea are kept at 19° C. insterile water filtered on activated carbon/ceramic cartridge and on 0.22μm Millipore filter membrane, without antibiotics. The water is changedevery two to three days in order to eliminate waste such as planarianwaste. The planarians are fed calf liver once a week. They are deprivedof food 1 week before the experiments to purge them. Their averageweight is 2.2 mg/worm.

1.2 Stem Cells:

Human dental pulp stem cells (CLS 300702) are maintained under standardin vitro culture conditions in DMEM/F12 (GibCoBRL) cell culture mediumsupplemented with foetal calf serum (10%) and antibiotic compounds suchas penicillin and streptomycin. (5000 U/mL, 5000 μg/mL respectively), ata temperature of 37° C. under a 5% CO₂ atmosphere. The cells are splitonce a week, after trypsinization (0.05%) to loosen the adherent cells,by a 1:3 dilution in DMEM/F12 culture medium (GibCoBRL) supplementedwith foetal calf serum (10%) and an antibiotic cocktail(penicillin-streptomycin), gentamicin, vancomycin, ciprofloxacin) (5000U/mL, 5000 μg/mL, 50 μg/mL, 10 μg/mL, 20 μg/mL respectively), aftertreatment with trypsin 0.05%, (GibCoBRL).

EXAMPLE 1 Preparation of the Planarian Extract (Protocol A)

1a) The planarians (25 individuals) are taken and washed twice with PBS(phosphate buffered saline, 1×) at pH=7.2, at room temperature, thenplaced in 550 μL of water (extract A1) or PBS (extract A2) or DMEM/F12(GibCoBRL) culture medium (extract A3), at a concentration of 110 mgplanarians/mL or 50 planarians/mL, at 4° C.

The planarians are ground with a plastic pestle, then the extract ispassed 10 times (in and out) through a 23 G syringe to break up thecells, the ground debris being between 5 and 10 μm in size.

1b) The extract is then centrifuged at 200 g for 5 minutes, at atemperature of 4° C.

The centrifuged extract then has two phases, a dense phase at the bottomof the tube and a supernatant aqueous phase. The supernatant aqueousphase is then collected (about 500 μL) and stored frozen at atemperature of −80° C., or frozen in liquid nitrogen (−196° C.), as a100 μL aliquot before use.

EXAMPLE 2 Preparation of Lyophilized Planarian Extract (Protocol B)

An aqueous extract of centrifugation supernatant of ground planarianscontaining culture medium, frozen are prepared according to protocol Aabove, is placed in a 2 mL glass lyophilization flask with stopperplaced at a temperature of −20° C. overnight before lyophilization.Lyophilization is carried out in a COSMOS freeze-dryer from the companyCRYOTEC (France) overnight. The lyophilisate is then stored at +4° C. inthe dark until use. Before use the lyophilisate is resuspended in 500 μLsterile distilled water.

2. Cell Regeneration Results.

EXAMPLE 3A Incubation of Human Stem Cells with the Aqueous ExtractObtained According to Protocol A

Centrifugation supernatants of ground planarians of the species S.mediterranea obtained in step 1b) of Example 1 are added at differentfinal dilutions of 1:5, 1:20 and 1:100 to human dental stem cells at5×10³ cells/well, of a 48-well plate, cultured in DMEM/F12 medium(GibCoBRL) supplemented with foetal calf serum (10%) and penicillin andstreptomycin (5000 U/mL, 5000 μg/mL respectively), at 37° C. and under5% CO₂ atmosphere. The cells are then maintained under the followingconditions: 37° C., 5% CO₂ atmosphere for 9 days. After 9 days the cellsare counted and the growth determined.

FIGS. 1A to 1C show the growth of human stem cells treated with extractsA1 to A3 diluted 1:5 (FIG. 1A), extract A3 (with culture medium) diluted1:20 (FIG. 1B), and extract A3 (with culture medium) diluted 1:100 (FIG.1C), for 9 days. (*p<0.05). The relative cell multiplication ratio wasquantified on the y-axis compared with a control with no extract ofground material.

FIG. 2 shows the growth of human stem cells treated with extract Acontaining culture medium (A3) diluted 1:20 for 9 days (A2) and controlbefore treatment (A1). The cells are stained with Giemsa.

A maximum increase with more than 50% increase in the number of cells isobserved in the presence of extract A3 containing culture medium diluted1:20 compared with the control culture without extract according to theinvention, whereas for the 1:5 and 1:100 dilutions the growth is lessthan or equal to the control.

A maximum increase with more than 40% increase in the number of cells isobserved in the presence of the aqueous extracts with water alone (A1)and containing PBS (A2) diluted 1:20 compared with the culture controlwithout extract according to the invention.

On the other hand, FIG. 3 shows the absence of growth on day 9 of humancells treated with uncentrifuged ground whole planarians, i.e. not freeof solid debris obtained in Example 1a).

EXAMPLE 3B Incubation of Human Stem Cells with Lyophilized PlanarianExtract (According to Protocol B)

The lyophilisate (B) is resuspended in 500 μL of water, then added at afinal dilution of 1:20 to human dental stem cells at 5×10³ cells/well,of a 48-well plate, in culture in DMEM/F12 medium (GibCoBRL)supplemented with foetal calf serum (10%) and penicillin andstreptomycin (5000 U/mL, 5000 μg/mL respectively), at 37° C. and under5% CO₂ atmosphere. The cells are then maintained under the followingconditions: 37° C., 5% CO₂ atmosphere for 9 days.

After 9 days the cells are counted and the growth determined. After 9days, an increase in the number of cells in the presence of extract B ofabout 27.7% is observed as shown in FIG. 4.

FIG. 4 shows the growth of human stem cells treated with extract Bdiluted 1:20 for 9 days. (*p<0.05) The relative cell multiplicationratio was quantified on the y-axis compared with a control withoutextract of ground material.

EXAMPLE 4 MALDI-TOF Mass Spectrum of the Planarian Extract

Mass spectrometry is a known physical analytical technique for detectingand identifying molecules of interest by measuring their mass. Itsprinciple lies in the gas phase separation of charged molecules (ions)according to their mass/charge ratio (m/z). The applications of massspectrometry are very broad and mainly concern the identification ofpeptides or proteins, the analysis of their amino acid sequence or thedetection of post-translational modifications.

Mass spectrometry in its MALDI-TOF (matrix-assisted laser desorptionionization-time-of-flight) variant has established itself in biologicallaboratories in recent years. Indeed, its advantages are numerous: (1)speed of identification (in a few minutes); (2) accuracy ofidentification (without the need to know the type of microorganismsought); and (3) low cost per sample (less than 50 cents per sample).

Among the mass spectrometer manufacturers, two complete MALDI-TOFsystems are available for sale, marketed by Bruker Daltonics (Germany).

1. A sample of aqueous extract with water alone A1 diluted 1:20 ofExample 3A was deposited on a HCCA matrix, trichloroacetic acid, water,and acetonitrile. The analysis is performed on a Microflex LT10MALDI-TOF mass spectrometer. The results are collected with the MALDIBiotyper RTC software.

One μL of u supernatant is deposited on the target plate of thespectrometer and dried for 10 minutes. One μL of saturated HCCA matrixsolution is added on top, dried for 10 minutes, then a mass spectrum ofthe protein extract is made on a Microflex™ LT (Bruker).

2. The spectrum acquisition method includes the following steps andfeatures:

introduce the plate (Microflex target reference model 224989 known asMSP 96) containing the deposits into the Microflex LT mass spectrometer,

start the computer associated with the spectrometer to start theanalyses, and

launch the acquisition of the MALDI-TOF mass spectra.

The parameters applied to the mass spectrometer are as follows:

positive linear mode, Electrode IS1: 20.00 kV, Electrode IS2: 18.05 kV,Focusing electrode: 6 kV, Laser frequency: 60 Hz, Detector gain: 8.8×,Post-ion-extraction (PIE): 120 ns, Mass range: 700 to 20000 Da.

The spectra are obtained by an automatic method controlled by theFLEXCONTROL® software from the manufacturer BRUKER DALTONICS® and theparameters for each spot are as follows:

serial numbers of 40 shots: 6 on different positions of the spot(6×40=240 spectra),

so that the shots have scanned the whole sample homogeneously, theacquisition is made on 6 different positions according to a hexagonalgeometry,

pre-shots: 10 shots at 40% of the maximum laser power are used todesalinate the sample (contamination by mineral salts),

laser power: 30% to 45% regulated via the FLEXCONTROL® software,

spectrum selection: over a mass range of 2000 to 20,000 Da.

The acquisition of the spectra is finally carried out via the BIOTYPER®software from the manufacturer BRUKER DALTONICS® which allows theautomatic method described above to be applied to a series of samplestaking into account the criteria mentioned above and a quality score orvalidation score which represents the addition of 2 scores n1+n2 (n1being a function of the number of peaks and n2 being a function of thebackground signal/noise ratio):

n1 is the score assigned according to the number of characteristic peaksin the spectrum n, where: n1=0 for n≤8, n1=1 for 8<n≤27, n1=2 for27<n≤43, n1=3 for 43<n≤78, n1=4 for 78<n≤86, n1=5 for 86<n≤110, n1=6 for110<n, and

n2 is the score assigned according to the value of the signal-to-noiseratio (Rmax) of the peak of greatest intensity in the spectrum, withn2=0 for Rmax≤6.8, n2=1 for 6.8<Rmax≤26.8, n2=2 for 26.8<Rmax≤52.6, n2=3for 52.6<Rmax≤208.6, n2=4 for 208.2<Rmax≤398.6, n2=5 for398.6<Rmax≤1,092.2, n2=6 for 1,092.2<Rmax.

A spectrum is taken into account if its validation score (n1+n2) isgreater than 2.

The full spectrum is shown in Table 2 below, with the largest peaksgiven in Table 1 above.

In Table 2, the values given are average values over 3 extracts and 3measurements per extract. “SD” is the standard deviation.

TABLE 2 m/z S/N m/z (SD) S/N (SD) 8604.48 171.52 2944111.66 2325738.788558.14 103.75 3757582.77 2412169.59 8364.32 270.77 4299073.533953124.73 8231.72 370.12 6273650.35 4516995.49 8109.81 473.7014474095.01 11993621.48 8091.08 485.10 16468398.81 10785668.45 8066.99473.89 10995647.50 9275395.50 8040.23 453.94 5195376.26 413314.928028.26 448.60 1362266.53 564362.67 8007.05 458.07 1529078.69 723901.787977.47 449.60 1934246.85 606979.25 7956.26 430.41 3779700.78 2089476.267926.83 439.17 6343305.77 2204916.89 7915.14 445.50 5271199.783945913.58 7888.82 422.60 9915944.44 6394858.15 7845.95 451.048949566.35 8892715.74 7774.23 390.89 10047314.25 14751532.02 7766.87396.60 17998107.53 16324726.64 7739.54 417.36 6983093.88 4683065.737711.45 420.11 11638867.28 11162939.84 7682.37 447.41 2849076.4626573.96 7620.78 384.09 1522404.95 1571602.58 7603.31 366.14 3797954.75301700.94 7507.22 462.35 4988724.05 1053369.21 7460.86 447.47 3010910.15786290.91 7453.34 443.82 1473989.18 754971.29 7441.65 442.85 2268420.85265803.84 7428.84 436.23 990780.63 482569.27 7411.46 434.77 3113006.6544381.36 7379.53 413.93 2507654.97 237260.33 7357.69 419.02 3439236.211696465.36 7348.32 417.87 4953557.73 865045.30 7337.73 413.55 5789551.76904998.52 7327.41 406.71 1938361.88 100617.30 7298.87 418.81 8970671.1710923542.62 7291.71 416.32 15579329.31 11046360.56 7233.87 458.442108878.03 1112809.94 7224.58 467.28 2188902.66 1253603.56 7211.60462.63 1277388.33 46435.77 7183.03 474.47 10858543.09 6841857.42 7166.12461.61 6005883.09 4377180.05 7141.72 462.27 8454369.88 12773497.187127.32 462.44 15980137.91 13198728.24 7117.16 460.20 1943880.501297607.73 7098.11 474.95 28478914.80 34393160.51 7089.16 479.6240768433.01 42971633.85 7068.69 472.09 3149404.57 1069351.59 7057.38475.83 4015369.66 840859.73 7049.75 475.55 3557870.44 341332.63 7046.15475.01 3404514.08 572811.01 7029.85 469.40 16173381.98 27409009.497019.69 465.68 29617067.96 31933238.72 6999.99 447.67 10403410.494241581.40 6994.31 450.26 12077581.56 3743384.83 6971.95 439.3119264755.03 20608829.34 6958.98 433.46 7134836.63 5304127.76 6948.13440.32 9541839.49 15202317.68 6927.62 429.41 19426439.15 15239899.386905.24 428.66 8416940.87 2205389.06 6896.02 426.82 5501032.421797378.54 6873.20 404.03 5662071.47 988813.38 6862.21 407.24 5647756.06486820.14 6849.90 398.50 4232957.36 2380406.49 6843.00 401.23 2871706.788206.17 6831.95 396.09 11137649.21 7908745.51 6820.90 395.54 4224912.361165458.98 6805.35 398.01 4634248.86 2098831.45 6800.77 396.395973899.73 1989363.37 6782.34 393.37 19055281.15 21136742.86 6770.31391.57 35509917.83 13034885.15 6745.15 407.32 18571000.28 15262519.686725.36 408.71 8653775.85 4308862.55 6709.87 408.66 5238132.911539867.36 6700.89 409.86 7534629.94 2915163.71 6696.13 411.068830295.99 3256750.86 6680.99 407.15 4231387.69 593937.32 6654.14 430.443521998.94 1238912.77 6640.58 422.93 3211633.91 246128.86 6625.94 423.151532349.37 792001.80 6615.85 426.74 6569850.42 2709202.72 6600.71 441.145420849.20 2607427.19 6581.25 440.63 4607565.27 5977204.13 6571.96433.59 8440513.66 5900474.08 6560.55 441.43 6770162.75 4190931.566548.88 443.03 8297991.19 897107.33 6539.51 441.18 1698707.19 918513.036520.81 429.87 4671420.38 784239.83 6510.06 437.54 11852564.161975111.53 6497.56 430.01 9963548.39 4648988.58 6486.81 435.5711018822.76 5444923.89 6478.80 431.50 10627202.29 7294336.61 6471.06431.57 6997871.01 6763605.29 6457.19 441.74 9893297.41 8332244.066453.29 438.94 3714855.88 283840.79 6442.34 432.99 13686239.4611824081.49 6430.23 428.59 5363664.11 3097124.33 6418.96 432.145051217.13 1408786.51 6406.03 428.78 4700220.72 4284253.70 6401.90428.42 5939522.66 4703337.99 6387.38 431.14 4660595.31 684934.44 6376.39431.18 4951125.32 1356190.69 6362.96 431.07 2672270.13 1511767.266355.85 431.23 6478221.94 2029795.02 6337.93 429.77 14364514.8813011112.00 6334.17 429.91 23516628.10 10002697.44 6315.15 419.8253493918.67 60231591.15 6301.95 415.75 4271746.45 1331550.38 6296.98415.38 7983195.65 3877555.77 6289.60 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81.3515139176.78 16312211.05 2795.32 73.80 40385980.59 3023772.39 2789.9274.46 13714579.43 2484950.91 2783.54 76.49 13128060.30 7235481.092778.50 79.56 20025295.07 17769176.18 2773.95 78.42 31978467.5214828688.66 2768.56 82.01 13971814.33 8845816.58 2762.30 80.6713479776.84 6737948.36 2759.05 79.72 18220985.01 503018.26 2755.16 81.282669732.32 2608631.65 2744.14 86.48 66874118.58 4951549.34 2736.14 86.9715227619.88 23217364.19 2729.24 83.67 40047790.36 14621368.10 2721.7886.47 32494845.54 9861038.35 2715.02 84.99 15566052.72 8014560.312708.50 86.17 32274863.45 21064023.15 2703.23 89.34 17707662.869238787.31 2693.93 87.00 24576409.78 14267235.68 2688.55 86.7133478516.12 13966490.89 2682.50 85.54 7418656.27 7893251.31 2680.1386.01 14684559.64 5040338.06 2674.98 85.99 17270487.71 19359469.432671.23 84.33 26727133.13 22398149.03 2665.67 88.83 5283789.073865732.85 2658.27 88.08 25273120.09 4393075.10 2653.65 89.1621547519.99 10611230.68 2649.35 85.70 13122469.10 8028955.22 2644.1687.47 10781319.39 1787055.71 2640.07 88.57 11736706.58 8250586.672636.65 89.85 10649457.50 6282520.10 2632.87 86.40 15703800.979817664.30 2626.10 90.30 19714480.57 7192677.53 2617.47 85.4331226146.63 12152168.58 2610.05 79.00 41708370.08 2574178.30 2600.8083.03 36082461.25 13946996.68 2592.59 78.32 26693374.13 16492181.962585.54 82.28 27921741.92 2302322.65 2578.10 81.89 15505572.8921037839.04 2571.80 79.51 34182656.60 14866566.57 2566.30 81.5810833201.94 11266461.17 2560.42 82.35 34069769.06 32034403.31 2552.2275.43 3378399.87 2644712.96 2547.93 76.08 39196474.09 11566189.712538.71 75.47 10921385.07 7323148.51 2530.88 76.21 49198701.67 495839.332520.43 72.41 16925388.39 4190562.13 2512.78 78.61 27034734.4919178045.09 2507.07 76.66 12105670.72 7318551.29 2503.62 75.648231720.99 4926449.30 2501.89 75.25 2695697.87 742949.18 2496.47 74.0545085195.33 2401171.89 2486.16 71.12 11333343.46 6507735.71 2481.8070.62 21497226.33 978699.11 2476.14 74.41 25065131.93 2427967.69 2467.2870.91 13698532.63 16366245.31 2464.08 69.23 19854347.56 18973328.272456.24 70.64 16426566.99 16222940.71 2451.65 73.30 15780379.699008397.92 2447.90 72.24 17762250.78 7401549.72 2440.34 73.8822477712.86 13681518.10 2437.27 72.67 33473985.92 6001138.09 2430.2170.83 9896984.85 6482298.70 2422.80 71.42 20767328.32 12227382.262414.26 77.06 36194417.10 7221990.85 2410.11 76.89 13225126.186849103.58 2404.84 80.56 15972011.56 15761214.35 2397.44 79.1342447972.69 3116361.06 2389.44 81.77 12312179.10 7824745.27 2387.2382.96 7638563.78 3042873.54 2384.36 80.57 6792608.82 1830983.51 2377.0081.79 19979882.56 8784863.39 2373.87 82.50 19391076.68 9047207.392367.94 80.35 10842751.05 1338523.23 2358.39 84.95 67605624.6916696518.60 2346.18 73.79 17915481.75 6782703.98 2340.73 77.9413405582.97 16646151.32 2338.52 77.42 20400378.89 20392483.90 2332.7476.88 12762647.74 8192561.47 2330.07 76.14 3695080.60 3155341.88 2329.2275.86 5267830.78 3066580.05 2323.74 72.19 11773784.40 16320386.962317.52 73.83 34610806.46 19678295.79 2311.99 73.46 38049986.5822383013.79 2307.60 75.80 21589527.00 2465183.16 2301.23 77.849510428.47 8605995.62 2296.66 76.30 24513429.85 3249221.38 2292.96 75.743722069.07 3539327.35 2287.59 77.52 33697641.24 27030677.63 2279.4082.47 33667781.63 25102629.42 2276.00 82.95 51327515.38 20485990.222263.79 74.98 19259319.45 19184327.67 2262.77 75.04 34737064.859498311.75 2251.65 77.49 22268464.86 16528203.81 2249.40 77.6030879684.57 18577805.62 2239.40 78.95 12661031.88 7926297.90 2234.2577.38 21016522.69 10555362.43 2230.89 76.54 28617627.12 81636.22 2224.3376.96 13832527.48 7198557.38 2222.02 76.31 12418154.19 12094252.832216.52 78.35 16600502.80 9176382.93 2213.54 79.17 16931528.3211801163.54 2207.17 77.82 26738129.67 10901707.68 2204.60 79.1821147827.70 21699405.87 2198.23 75.24 16466096.25 6976682.27 2192.9971.51 8450148.18 6644247.22 2189.93 72.42 15984063.74 15914916.092186.73 71.56 23277490.55 17871975.88 2180.00 75.57 17250213.669016909.20 2174.61 78.38 19066625.56 18471707.30 2171.39 81.1316727577.51 14878434.86 2167.57 81.79 17486637.04 16089300.86 2160.3481.30 55117901.61 60827538.28 2149.22 90.56 7330904.48 3880842.062148.05 90.60 10061016.94 1428645.33 2142.13 90.49 17187131.1515201450.97 2138.95 89.15 27732772.22 12816866.81 2132.64 94.0723160451.34 7451209.21 2129.20 92.91 19895001.30 15037326.02 2124.3293.49 21647446.88 5014265.55 2118.49 90.68 31583760.09 39696.62 2112.6593.18 23618680.16 7816955.71 2103.87 88.87 19067735.65 12282181.232101.18 88.82 27141754.05 5110877.70 2093.21 85.81 30968672.4525786526.05 2087.98 81.89 7652250.80 2778589.51 2084.72 84.5230340667.85 26243240.46 2079.30 88.75 23336380.16 10191775.32 2074.4487.27 4413018.54 975274.21 2072.69 87.67 3599873.96 2013970.86 2070.5588.16 17342208.42 16992808.57 2066.72 90.28 2780537.52 1703714.492062.82 88.08 9287962.27 1210143.08 2060.27 88.30 15994010.0614591780.36 2055.54 84.71 7328859.22 2561419.94 2048.65 80.2531398129.93 22701765.90 2045.69 79.37 12548381.88 2322244.68 2039.2378.24 45574115.58 25496143.29 2027.74 74.49 29120916.22 18892121.622023.66 74.48 30730437.22 20507268.28 2016.77 73.36 16975844.498032399.88 2014.17 72.69 20494142.08 1388076.84 2008.18 69.7714062256.07 1918569.47 2004.98 69.01 7766693.94 1726234.57 2001.38 67.1717555377.17 13683669.28 1996.53 67.96 15183356.16 17159307.34 1994.0868.55 20721744.25 18039134.48 1984.69 70.97 32665114.41 6245320.601978.14 73.80 28168134.15 306843.58 1972.58 71.67 9983946.70 3454559.411969.84 71.69 17353347.62 13486732.22 1962.66 71.16 31890470.6528696808.33 1955.18 66.40 19687405.65 17400459.78 1952.66 65.9729178846.18 18940406.32 1949.44 64.51 16979591.33 10075425.44 1944.1666.79 15523473.36 6258297.22 1940.78 65.95 5705294.62 5019942.34 1939.0866.51 4150369.47 1204834.73 1934.52 65.88 50279169.44 7135733.27 1927.9468.30 19603220.48 16744393.40 1923.21 72.87 5881498.34 202589.62 1920.2074.15 9083859.30 4310570.91 1917.28 72.06 17377661.61 2139713.46 1911.1476.77 27008387.97 22092224.62 1905.91 76.89 31224136.45 32602387.721900.23 78.73 27662460.12 14202731.43 1895.79 80.81 20288691.3918805809.02 1890.31 80.58 18507274.18 11899128.80 1884.50 82.7923273916.64 14086832.57 1881.14 84.43 21021152.50 16986700.93 1876.6982.45 15323751.91 9112165.05 1872.80 84.84 29895119.73 13106557.121866.47 83.47 14095096.03 8664460.73 1861.14 84.13 18284073.9511297956.75 1859.15 84.16 21472881.03 7119960.92 1854.31 83.1318717665.33 12336629.53 1852.64 82.86 24657935.23 12935523.67 1847.6081.29 15686493.65 9901435.51 1842.79 77.92 10704277.55 10762227.081837.79 79.10 27527289.47 12018037.66 1834.07 80.25 7654986.216806629.36 1830.92 81.94 17281665.93 7176581.34 1824.93 83.2725318012.89 27057360.22 1820.45 83.73 52492545.65 13559729.17 1813.0780.85 14872603.40 1901051.15 1806.94 78.07 45318420.43 16101971.731801.94 78.70 7913796.27 2410831.89 1799.52 78.44 13142802.41 4186108.741795.30 78.75 29968728.66 21196423.90 1790.63 75.78 41172977.0419064114.05 1779.38 67.93 40688660.05 46508273.27 1776.74 66.562962514.49 724516.02 1774.33 65.29 18187824.69 17753519.51 1770.69 63.2331793423.47 28319595.26 1762.17 56.32 13179813.83 14877684.69 1758.6757.13 66708798.32 38215140.29 1751.64 57.49 34429398.99 26114382.081747.24 54.79 36285932.62 37703784.71 1739.88 61.78 6964022.434600629.08 1737.19 62.29 7697357.10 4052505.50 1734.43 60.71 21985181.3712736599.05 1729.02 57.69 23882756.92 8858907.53 1721.57 52.1450870806.45 49797820.46 1712.46 45.10 24263580.92 15186916.98 1706.2645.02 30511773.52 15564404.48 1700.78 43.54 28016393.48 14108489.571691.13 36.63 35108988.32 35671765.16 1684.97 33.17 32720714.5629517546.61 1681.39 31.64 40372346.30 37580574.64 1677.66 31.0310083221.90 9842889.10 1674.82 32.41 14077675.84 3264717.85 1670.1829.22 38364245.24 28421815.74 1661.53 25.56 35186159.98 12526902.941657.27 22.51 21962778.79 22758040.35 1646.81 20.91 39561118.209611280.36 1639.69 17.75 70362502.17 19800253.72 1623.91 23.9471989697.44 77097377.73 1616.75 28.13 18078945.11 6801902.60 1613.7328.51 12601310.15 11732186.68 1608.26 29.70 22665641.25 7346444.491603.34 28.69 30073688.45 5063255.00 1600.06 28.50 19817910.1816742721.02 1594.77 25.19 29767782.34 12651483.21 1591.46 26.1925160053.37 14053755.90 1587.81 23.15 32740069.22 7752483.37 1577.5027.34 44155964.40 22113236.10 1574.17 29.27 2618412.92 2739445.251572.48 29.96 8283974.26 435382.92 1567.70 30.87 35268646.70 27272113.561563.74 32.74 57311974.32 16437414.62 1554.30 25.87 38180847.239009048.90 1544.06 26.58 40809046.72 10263841.48 1539.87 26.5931718484.43 17607978.22 1535.18 28.11 46717446.35 33662799.97 1529.1031.37 15159581.47 22717428.35 1527.41 31.23 44339825.44 5458202.031518.83 32.72 23140434.72 11530542.73 1513.63 34.93 22581528.4019821543.03 1510.00 36.67 13460381.12 3185724.52 1507.90 36.0122338977.67 14662503.91 1504.83 38.62 37857106.76 7398153.07 1498.3034.99 23455026.94 14999137.06 1471.78 10.77 154656464.21 139271192.431458.56 0.93 90080868.28 40953029.86 1448.81 0.79 40436778.1710521402.98 1446.08 1.39 5336848.90 4681017.31 1442.99 3.38 29626710.3231254997.18 1436.03 5.97 72212769.22 12704723.32 1425.05 11.0748305773.28 25252135.06 1420.39 10.81 44522025.94 45324620.71 1414.1410.10 38770175.08 58152491.82 1407.93 4.65 77562603.59 55895325.591403.12 1.24 35073914.18 12491904.33 1388.74 4.16 89732035.5811915766.84 1381.90 1.94 36924967.60 30283407.95 1380.40 1.3134664243.09 35268757.89 1374.98 6.55 3189606.98 756150.71 1369.88 10.3932520115.84 27236740.14 1366.68 11.29 43834473.71 30662758.00 1358.087.28 59090476.39 36152386.44 1351.25 8.21 74618701.10 37123567.081344.03 3.30 26923014.64 27446679.17 1338.35 3.56 45757983.03 3986455.481333.94 5.01 39154116.34 8667291.47 1327.25 3.25 35680822.22 3853203.881323.35 2.86 37751869.64 2452787.33 1320.95 0.49 34482975.59 1749809.661316.80 1.37 10396138.14 6810361.34 1312.93 1.86 53676390.81 25869702.201306.92 6.48 47506102.23 1971607.94 1300.99 6.45 28227492.69 9821166.981299.72 6.83 31811940.40 8197514.94 1294.91 7.21 20838316.31 11141120.381289.17 6.20 42867890.50 18552533.14 1285.04 4.27 49059615.1914248882.44 1282.00 2.75 41860201.65 32757873.62 1275.82 2.6735067732.33 31922106.43 1273.99 2.30 47317853.29 41105587.82 1268.374.66 19566412.02 16320587.04 1259.12 8.59 109923932.10 18133902.921252.95 6.76 54738932.66 60212880.81 1247.56 1.60 14417418.5913735264.29 1242.22 2.95 41553114.16 28582701.00 1239.26 2.5257398324.08 18729637.28 1234.34 4.59 53774436.49 34118311.02 1228.210.24 83321749.38 12519601.00 1221.31 1.67 63564527.51 31521726.741216.96 3.52 60178620.27 24339697.06 1210.51 2.35 36346810.13 7334799.471208.30 1.32 36004719.04 13099228.96 1205.31 3.28 21179465.98 9588045.221200.84 6.47 61510491.41 32154185.38

1. An aqueous extract useful notably for the regeneration of human ormammalian animal cells, comprising an aqueous extract free of anyinsoluble solid debris of ground organisms of the Platyhelminthes typehaving a regeneration capacity, said organisms being aquatic flatwormsof the planarian type which are non-parasitic and non-pathogenic inhumans or mammalian animals, said aqueous extract containing at leastthe intracellular components of the cells of said organisms of a celllysate of said cells of said whole organisms.
 2. The aqueous extract asclaimed in claim 1 characterized in that said organisms are of thespecies Schmidtea mediterranea.
 3. The aqueous extract as claimed inclaim 1 characterized in that said aqueous extract of ground materialcomprises a centrifugation supernatant of said ground material.
 4. Theaqueous extract as claimed in claim 1 characterized in that said aqueousextract of ground material further comprises a cell culture medium,preferably a culture medium capable of culturing human or animal cellsto be regenerated.
 5. The aqueous extract as claimed in claim 1characterized in that said aqueous extract of ground material comprisesthe grinding of 50 to 200 mg worms per mL aqueous solution; preferably75 to 150 mg worms per mL aqueous solution, said aqueous extract ofground material being further diluted preferably more than 1:5 and lessthan 1:100, preferably diluted between 1:10 and 1:50.
 6. The aqueousextract as claimed in claim 5 characterized in that said aqueous extractof ground material comprises the grinding of 110 mg worms per mL aqueoussolution; said aqueous extract of ground material being further diluted1:20.
 7. The aqueous extract as claimed in claim 1 characterized in thata spectrum of said preparation by MALDI TOF mass spectrometry comprisesthe 16 characteristic majority peaks mentioned in the following Table 1,wherein the individual characteristic peaks are numbered in the firstcolumn, and the following parameters have the following meanings: M/z:ratios (mass in daltons/charge) on the x-axis of the differentcharacteristic peaks of the spectrum, S/N: ratios (peakintensity/background noise intensity) of the different characteristicpeaks of the spectrum, TABLE 1 M/Z S/N standard standard M/Z deviationS/N deviation 1 8618.401 118.031 10852424.610 11358052.088 2 8270.340143.008 4620996.274 5564954.057 3 7696.982 133.512 5240359.9237010776.551 4 7236.251 151.442 8121705.361 11556323.264 5 6803.343116.896 10708147.034 18373045.879 6 6261.480 163.376 11319617.93813604702.931 7 5790.573 116.345 21402383.693 37624416.818 8 5330.950180.245 23085485.049 21438811.717 9 4787.963 120.365 22731248.66823171439.149 10 4298.636 175.285 24470357.501 21953618.321 11 3793.559113.425 21664847.045 17673326.341 12 3282.861 172.508 24634530.73627003268.283 13 2792.441 122.205 22551669.576 22432014.766 14 2294.281174.311 20589324.299 17568023.101 15 1807.544 115.976 19890585.11418182405.582 16 1395.545 119.208 33019669.177 29193485.572.


8. A lyophilisate of an aqueous extract of ground material as claimed inclaim
 1. 9. A process for preparing an aqueous extract as claimed inclaim 1 characterized in that the steps are carried out in which: a)said living organisms, preferably washed with water and not fed for atleast one week, are placed in an aqueous liquid medium containingpreferably a cell culture medium; b) the aqueous preparation obtained instep a) is ground until lysis of the cells of said organisms isobtained, preferably the ground material debris being between 5 and 10μm in size, c) insoluble debris is removed by centrifugation; and thesupernatant is collected, and d) preferably, the centrifugationsupernatant is diluted in an aqueous solution more than 1:5 and lessthan 1:100, preferably diluted between 1:10 and 1:50, said solutioncomprising preferably still components of a cell culture medium capableof culturing human or animal cells to be regenerated.
 10. Use of anaqueous extract or lyophilisate as claimed in claim 1 for themanufacture of a composition useful for regenerating human or mammaliananimal cells.
 11. Use of an aqueous extract or lyophilisate as claimedin claim 10 for the manufacture of a composition useful for regeneratinghuman or mammalian animal stem cells.
 12. Use of an aqueous extract orlyophilisate as claimed in claim 10 in combination with suitablecarriers and/or excipients for the manufacture of a food, cosmetic ordermatological composition.
 13. A cosmetic composition with ananti-ageing effect on the skin comprising an aqueous or lyophilizedextract as claimed in claim 1 and excipients suitable for topicalapplication to the skin of said composition in the form of gel, lotion,cream, ointment or soap.
 14. Use of an aqueous extract or lyophilisateas claimed in claim 1 for regenerating human or mammalian animal cells.15. Use of an aqueous extract or lyophilisate as claimed in claim 10 forregenerating human or mammalian animal stem cells.